1. Field of the Invention
This invention relates to an assay method for performing a qualitative analysis or a quantitative analysis of a sample, which contains an analyte (i.e., a substance to be analyzed), with a high sensitivity.
2. Description of the Related Art
Recently, there have been proposed various simple devices for developing test body solutions and detecting analytes contained in the test body solutions, and various kinds of devices for detection of external diagnostic medicines, poisons, and the like, are available commercially. Examples of the aforesaid devices include the devices utilizing immunochromatographic techniques. With the immunochromatographic techniques, large scale facilities and equipment are not required for making judgments and analyses, and operations are easy to perform. Specifically, in the cases of the immunochromatographic techniques, the results of the analyses are obtained with processing wherein, after a test body solution having the possibility of containing an analyte has been spotted onto a support, the test body solution having been spotted is merely allowed to stand for a period of time falling within the range of approximately 5 minutes to approximately 10 minutes. Therefore, the immunochromatographic techniques have widely been used as the judgment or analysis techniques, which are simple and quick and have high specificity, in a wide variety of use applications, such as clinical examinations in hospitals and assay tests in research laboratories.
Ordinarily, physiologically active substances or environmental pollution substances, such as natural substances, toxins, hormones, or agricultural chemicals, act upon organisms only with markedly small quantities which are not capable of being detected with the conventional ordinary immunochromatographic techniques. Therefore, nowadays there is a strong demand for an immunochromatographic technique with which the aforesaid substances are analyzed quickly, in a simple manner, and with a high sensitivity. Accordingly, besides the immunochromatographic techniques having the simple processing for spotting a test body solution, which contains an analyte, onto a support, and thereby detecting the analyte, for example, there have been used the immunochromatographic techniques wherein, after a test body solution containing an analyte has been spotted onto a support, and the analyte has thus been fixed to the support, the support is washed with a washing liquid, wherein a reaction substrate liquid, an amplifying solution, or the like, is brought into contact with the analyte having thus been fixed, and wherein a signal obtained from the analyte is thus amplified and detected.
As an example of the immunochromatographic technique having a high sensitivity as described above, an immunochromatographic technique is disclosed in, for example, International Patent Publication No. WO93/03176. The disclosed immunochromatographic technique comprises the steps of: causing a test body solution, which contains an analyte and has been brought into contact with an enzyme-labeled antibody, to flow with respect to a solid phase support, to which an analyte capturing reagent has been bound, the analyte-enzyme-labeled antibody complex being thereby bound onto the support, causing a washing liquid and an enzyme substrate liquid to flow in a direction reverse to the direction of the flow of the test body solution, an analyte-enzyme-labeled antibody-enzyme substrate complex being thereby formed, detecting a signal, such as coloring, occurring due to the enzyme substrate, and thereby analyzing the analyte.
With the immunochromatographic technique disclosed in International Patent Publication No. WO93/03176, the signal obtained from the analyte is amplified by the enzyme substrate. Also, by the reversible flow performed such that the washing liquid and the enzyme substrate liquid are caused to flow in the direction reverse to the direction of the flow of the test body solution, the contact of the analyte capturing reagent, which has been bound to the support, and the analyte-enzyme-labeled antibody complex with each other is enhanced. Therefore, the analysis is capable of being made with respect to the trace quantity of the analyte.
However, with the immunochromatographic technique disclosed in International Patent Publication No. WO93/03176, wherein the washing liquid is caused to flow over the entire area of the flow path through which the test body solution has flowed, the problems arise in that a large quantity of the washing liquid is necessary in order for a sufficient washing effect to be obtained. Also, the washing liquid and the enzyme substrate liquid are caused to flow in the direction reverse to the direction of the flow of the test body solution, i.e., from the downstream side with respect to the flow of the test body solution. Therefore, an unbound enzyme-labeled antibody and an unbound analyte, which have not been bound with the analyte capturing reagent on the support, are caused by the washing liquid and the enzyme substrate liquid to flow again on the support. Accordingly, limitation is imposed upon the detection with a high accuracy and a high sensitivity.